¿STUDY OF THE BEHAVIOR OF LACTOCOCCUS LACTIS AND LISTERIA MONOCYTOGENES IN COMPETITION FOR FOOD SAFETY IN DAIRY PRODUCTS¿

Microbial competition is a mechanism that occurs when two or more microbial species compete for ecological niches to support their survival and growth. Different factors can contribute to the outcome of microbial competition, such as molecules exchanged between the competing organisms for the regulation of cell densities and the initial spatial configuration of the microbe\u2013microbe interaction. Specifically, production of compounds that kill or limit the growth of competing strains or species can promote niche monopolization [2]. The released compounds include secondary metabolite antibiotics, bacterial peptides or low-molecular-mass organic compounds. The same happens in food, and it could be possible to explore this 'molecular' communication to improve food safety. In that sense, it is very important to develop tools in the control of bacterial species for a better food safety. The present work focused on the study of microbial competition between Listeria monocytogenes (LM) and Lactococcus lactis (LAC) monitored through proteomics, peptidomics and metabolomics approach. We study the secretome of these two microorganisms (Listeria monocytogenes and Lactococcus lactis) alone and in co-culture. In particular, we studied by proteomic analysis the evaluation of proteins secreted by bacteria through one/two-dimensional electrophoresis coupled to mass spectrometry (MALDI TOF). Furthermore, in order to characterize each secretome, label free Shotgun analysis was conducted using nano UPLC-MS system. Furthermore, the secretome of these microorganisms has been studied through first an untargeted proteomics analysis in vitro, followed by validation directly in a system resuming cheese. The objective of the last part of the project has been the monitoring of bacterial competition between through a combination of microbial Imaging mass spectrometry and LC-MS/MS, in order to investigate the metabolic profile of each bacteria in the interacting microbial colonies. In according with obtained preliminary data (one-dimensional and two-dimensional electrophoresis), new data highlighted, during competition, the higher production by Listeria monocytogenes of moonlighting protein Enolase (C1KY94) and Glucose 6 Phosphate isomerase (Q71X61), of Septation ring formation regulator EzrA (B8DHE7), involved into cell replication in regulatory mechanisms of cell energetics or metabolism and the lower secretion Endopeptidase P60 (P21171), protein associated with the cell surface and involved in the process of invasion. In parallel, L. lactis produced higher amounts of Page 6 of 114 Secreted 45 kDa protein and switched from lantibiotic Nisin A production to Nisin Z production. In competition with LM, LAC strain investigated produce higher amounts of Secreted 45 kDa protein with peptidoglycan lytic activity and the selective secretion of Nisin Z probably to improve lantibiotic solubility in less acidic environment. Lastly, IMS analysis revealed several interesting compounds during interaction of microbial colonies. At least six compounds are uniquely expressed during the interaction between LM and LAC. Among these, we focused our attention on three compounds: Cyclo-(Leu-leu), Cyclo-(Phe-Tyr), Cyclo-(L-Phe-L-4-Hyp). These compounds are cyclic peptides, isolated by Lactobacilli, with a biological activity]. In particular, they play an important role in bacterial cell to cell communication. Probably, these peptides have a role in inducing of the transcription of gene coding for Nisine. These results could be useful to setup new molecular strategies in the control of bacterial species for a better food safety

Reversible switch from hemoglobin A to C in sheep and recovery from anemia following experimental infection with Anaplasma ovis

Anemia causes a change in the type of circulating hemoglobin (Hb) in sheep carrying the βA-globin haplotype, where the Hb A is replaced with Hb C, unlike Hb B. The effect of the substitution of Hb A with Hb C on the recovery from anemia was investigated by comparing the hematological picture of sheep, following experimental infection with Anaplasma ovis. The blood values were obtained from 3 AB and 3 BB Hb sheep after the development of the disease where anemia is a pathognomonic symptom. The expression of the silent gene encoding for Hb C was detected by isoelectric focusing and quantified by high performance liquid chromatography. Both Hb AB genotype and Hb C occurrence were involved in the lower recovery from anemia in the trial

Peptidomics in veterinary science : focus on bovine paratuberculosis

Bacterial infections represent a serious burden both for animal production and human health (zoonosis). Faster and more reliable diagnosis are mandatory in order to avoid economic losses and antibiotics misuse. The development of new potential diagnostic strategies for the immunodetection of pathogens is closely linked to the discovery of small polypeptides with immunogenic or immunoreactive activity. The candidate peptides used for this purpose must have several properties principally represented by their specificity and their location in the bacterial cell. Both proteomics, peptidomics and bioinformatics represent powerful complementary tools to discover specific immunoreactive peptides useful for diagnosis or vaccine. Peptidomics of Mycobacterium avium subsp. paratuberculosis (MAP) represents a good example of the potential of this discovery-phase. This review reports a comprehensive update of the current scientific knowledge about proteins and peptides of MAP with already documented humoral response. These findings, together with bioinformatics tools available, could be extremely useful to design a better strategy for subclinical bovine paratuberculosis diagnosis. The knowledge provided also represents a reliable example on the workflow to be followed in the direction of the diagnosis of other diseases through a peptidomic approach

Serum proteomic profiles in CKCS with Mitral valve disease

Background: Myxomatous mitral valve disease (MVD) is the most common acquired heart disease in dogs, and the Cavalier King Charles Spaniel (CKCS) is the most studied breed because of the high prevalence, early onset and hereditary component evidenced in the breed. MVD has different severity levels, and there are many practical limitations in identifying its asymptomatic stages. Proteomic techniques are valuable for studying the proteins and peptides involved in cardiovascular diseases, including the period prior to the clinical onset of the disease. The aim of this study was to identify the serum proteins that were differentially expressed in healthy CKCS and those affected by MVD in mild to severe stages. Proteomics analysis was performed using two-dimensional gel electrophoresis separation and a bioinformatics analysis for the detection of differentially expressed spots. In a comparative analysis, protein spots with a p<0.05 (ANOVA) were considered statistically significant and were excised from the gels for analysis by MALDI-TOF-MS for protein identification. Results: Eight proteins resulted differentially expressed among the groups and significantly related to the progression of the disease. In mild affected group versus healthy dogs complement factor H isoform 2, inhibitor of carbonic anhydrase, hemopexin, dystrobrevin beta isoform X7 and CD5 molecule-like resulted to be down-regulated, whereas fibronectin type-III domain-containing protein 3A isoform X4 was up-regulated. In severe affected dogs versus healthy group complement factor H isoform 2, calpain-3 isoform X2, dystrobrevin beta isoform X7, CD5 molecule-like and l-2-hydroxyglutarate dehydrogenase resulted to be down-regulated. Complement factor H isoform 2, calpain-3 isoform X2, dystrobrevin beta isoform X7, CD5 molecule-like and hydroxyglutarate dehydrogenase were found to be down-regulated in mild affected group versus healthy dogs. All of these proteins except complement factor H followed a decreasing trend according to the progression of the pathology. Conclusion: The differential expression of serum proteins demonstrates the possibility these might be valuable for the detection and monitoring of the disease. Further longitudinal studies are required to determine whether differential protein expression occurs sufficiently early in the progression of the disease and with sufficient predictive value to allow proteomics analysis to be used as an early detection and on-line diagnostic tool

Intriguing hemoglobin polymorphism in Grey Alpine cattle and functional effect

Genetic diversity is important for the maintenance of the viability and the evolutionary or adaptive potential of populations and species. The hemoglobin (Hb) represent a unique system for studying adaptive changes because these oxygen-carrying proteins closely connect metabolic activities with external conditions. As far as cattle are concerned, previous works evidenced an unusual hemoglobin polymorphism in Italian Podolic cattle; particularly, the HBAY variant was found to be associated with decreased hematocrit and hemoglobin values. In addition, the presence of identical marker alleles in Podolic cattle (Bos taurus) and Indian zebu (Bos indicus) provided further support for the theory that Asian bovine genes could have contributed in the past to the Podolic breed. Aim: Based on the above-mentioned evidences, the present paper has a double aim: (i) to check the presence of the above variants in breeds belonging to different cattle groups, namely, two undoubtedly Brachiceros breed - such as Italian Frisian (IF) and Italian Brow (IB) - and Grey Alpine (GA), a rustic cattle breed for which there are evidences suggesting resemblance with breeds classified as Bos taurus primigenius; (ii) to confirm the functional effect of the HBAY phenotype. Materials and Methods: In this study, a sample of 79 unrelated registered GA cattle was selected from a recently-established herd located in the province of Bari (Apulia, Italy) composed by animals purchased over time directly from different breeders from Northern Eastern Italy and thus quite representative of the original breeding area. For comparison, 82 unrelated registered IF cattle and 92 unrelated registered IB cattle were sampled in 10 different Apulian farms. Hematological variables were evaluated using a hematology analyzer. Hb phenotypes were analyzed with isoelectric focusing in a pH range of 6.7-7.7 (PAGIF). A T-test for independent samples was adopted to evaluate the significance of the difference in the mean hematocrit and hemoglobin values between the group of animals displaying and not displaying the Y alpha-globin variant. Results: The positively charged Y alpha-globin variant, encoded by the HBA1Y gene, was observed only in Grey Alpine with a 0.13 frequency value as opposed to that of the alternative N variant. We also recorded the presence of the common variants A and B of the beta globin system, along with the silent mutant AZebu, the frequency of the corresponding HBBA, HBBB and HBBAZebu genes being 0.73, 0.23 and 0.04, respectively. Neither HBA1Y alpha gene or AZebu beta allele was detected in the other sampled breeds. Conclusion: Actually, the presence of identical marker alleles in IP and GA suggests they share a common origin, possibly tracing back to earliest ancestors such as Bos primigenius primigenius. In addition, the present study provides further support for a functional effect of HBAY as associated with decreased hematocrit values in cattle